The present invention generally relates to emulsifiers of biological origin, for use in numerous industries such as environmental management, health care, dental care, cosmetics and food product applications. The present invention further relates to a method for the production of said emulsifiers of biological origin, (herein after also called a xe2x80x9cbioemulsifierxe2x80x9d), using an esterase protein originating from the bacteria Acinetobacter, in combination with one of a variety of polysaccharides or biopolymers. The bioemulsifier composition thus obtained stabilizes a variety of oil-in-water emulsions. Since the bioemulsifier composition is versatile in regard to the biopolymer or polysaccharide used in the emulsification, many non-toxic polymers that are already widely in use, for example in the cosmetic or food industries, can be employed.
Bioemulsifiers are biological products that aid in the formation and stabilization of water-in-oil or oil-in-water emulsions. Unlike classical detergents whose activity is generally to reduce interfacial tension at the interface of two immiscible liquids thereby lowering the energy required to generate emulsions, amphipathic biopolymeric bioemulsifiers generally work by orienting themselves at the oil/water interface, in such a way that they interfere with coalescence of the oil droplets. This in turn stabilizes the emulsion, reducing the tendency of the droplets to coalesce.
Emulsifiers of biological origin are preferred over synthetic emulsifiers in instances where their use may have an impact on the environment, such as containment of oil spills, since bioemulsifiers are biodegradable, and are less likely to leave toxic by-products behind. In addition, bioemulsifiers may be relatively inexpensive to produce. U.S. Pat. No. 4,693,842 (Shilo) discloses a biopolymer purified from a cyanobacterium, which is useful in flocculation. U.S. Pat. No. 5,840,547 discloses a bioemulsifier named Alasan, originating from the bacteria Acinetobacter radioresistens strain KA53; and a method for its production.
There is previously disclosed in U.S. Pat. No. 4,395,353 (by the current applicant), a polymeric polyanionic, aminopolysaccharide, water-soluble bioemulsifier termed emulsan (molecular mass, 106) which is produced by an oil-degrading microorganism, Acinetobacter lwoffii RAG-1. Emulsan forms and stabilizes oil/water emulsions using a variety of hydrophobic substrates. The aminopolysaccharide backbone is composed of repeating subunits of a trimer consisting of D-galactosamine, D-galactosamine uronic acid, and bacillosamine (2, 4 diamino, 6-deoxy glucose). The amphipathic character of the biopolymer is due in large part to the presence of about 25% (w/w) acyl groups linked to the polymeric backbone in both ester, and amide linkages. The fatty acids vary in chain length from 2-18. In addition, a significant percentage of the fatty acids are hydroxylated (i.e. xcex2-hydroxy butyrate and xcex2-hydroxydodecanoic acid). During exponential growth the biopolymer is present on the cell-surface as a minicapsule and is released as a protein-polysaccharide complex as the cells enter stationary phase. Emulsan release is mediated by the action of a cell-surface esterase, which also constitutes one of the major proteins associated with emulsan. The non-covalently attached protein (between 15 and 25% by weight) can be removed from the polymer to generate a de-proteinated, partially active amphipathic bioemulsifier termed apoemulsan. The deproteinized apoemulsan is much less efficient in stabilizing very hydrophobic substrates. This hydrophobic activity can be partially restored by the addition of crude protein.
Emulsan technology was used as an industrial product in a host of cleaning and degreasing applications in ships, barges, storage tanks and various industrial environments. The U.S. Navy research labs have described the use of emulsan in the cleaning of sludge filters for all of their ships. In addition, emulsan was formulated for application in viscosity reduction of heavy oils and sludges by generating low viscosity homogeneous oil/water emulsions.
Emulsan is potentially toxic, since in crude form it is frequently contaminated with toxigenic lipopolysaccharide characteristic of all gram-negative organisms. Emulsan, therefore, must be highly purified in order to find uses in such applications as health care, dental care, cosmetics and food product applications.
It is the object of the present invention to provide novel amphipathic bioemulsifier compositions, which are non-toxic, versatile and relatively inexpensive to produce. Some of these compositions utilize non-toxic polymers that are already widely in use in various industries of commercial value such as the food and cosmetic industries, hence these polymers are considered both safe and inexpensive to employ. The novel compositions disclosed here allow emulsan technology to be more widely applied, since they are far more removed from the Acinetobacter bacteria than previously characterized emulsan compositions. Hence they need not contain contaminants typical of gram-negative bacteria.
It is also an object of the present invention to provide a method for producing the heresaid bioemulsifier compositions.
These and other objects of the present invention will become more apparent from the summary of the invention and the detailed description of the preferred embodiments, that follow.
The present invention relates to a bioemulsifier composition for forming and stabilizing oil-in-water and emulsions, comprising:
a) an esterase protein of 32.5 KD, wherein said protein is normally found in association with emulsan in the bacteria Acinetobacter, isolated from cell extracts from at least one strain of Acinetobacter; or recombinant preparations of said esterase protein isolated from esterase-producing vectors expressed in suitable hosts, or, peptide fragments of said esterase protein produced in any of a variety of means, including proteolysis or genetic cloning;
b) water-soluble polysaccharide polymer of any source, including bacterial, plant and synthetic sources; or biopolymers such as polyanionic heteropolysaccharides.
Further in accordance with a preferred embodiment of the present invention, the polysaccharide is selected from agarose, gum arabic, carrageenan, pectin, potato starch, xanthan, dextran, alginic acid, cellulose, chitin, colanic acid, ficoll 400, pullulan, polyvinyl pyrrolydone, stewartan, xylan or Acinetobacter calcoaceticus BD4 exopolysaccharide. It is appreciated, however, that any synthetic or biological polymer or polysaccharide may be employed as well.
Additionally in accordance with a preferred embodiment of the present invention, a stabilizer or a co-emulsifier can be added to the composition.
Still further in accordance with a preferred embodiment of the present invention, emulsan or apoemulsan are added to the composition, as a stabilizer or co-emulsifier.
Further in accordance with a preferred embodiment of the present invention, low molecular weight surfactants are added to the composition.
Moreover in accordance with a preferred embodiment of the present invention, the esterase protein is derived from Acinetobacter strain A. lwoffi RAG-1 and has the following amino acid composition:
Met lys phe gly thr val trp lys tyr tyr phe thr glu ser leu leu lys
Ala thr ile arg thr pro ser gln leu asn leu ala pro asn ala leu
Arg pro val leu asp gln leu cys arg leu phe pro gln asn pro
Thr val gln ile arg pro ile arg leu ala gly val arg gly glu glu
Ile lys ala gln ala ser ala thr gln leu ile phe his ile his gly
Gly ala phe phe leu gly ser leu asn thr his arg ala leu met
Thr asp leu ala ser arg thr gln met gln val ile his val asp tyr
Pro leu ala pro glu his pro tyr pro glu ala ile asp ala ile phe asp val
Tyr gln ala leu leu val gln gly ile lys pro lys asp ile ile ile ser gly
Asp ser cys gly ala asn leu ala leu ala leu ser leu arg leu lys gln
Gln pro glu leu met pro ser gly leu ile leu met ser pro tyr leu
Asp leu thr leu thr ser glu ser leu arg phe asn gln lys his asp
Ala leu leu ser ile glu ala leu gln ala gly ile lys his tyr leu thr
Asp asp ile gln pro gly asp pro arg val ser pro leu phe asp asp leu
Asp gly leu pro pro thr leu val gln val gly ser lys glu ile leu leu
Asp asp ser lys arg phe arg glu lys ala glu gln ala asp val lys
Val his phe lys leu tyr thr gly met trp asn asn phe gln met
Phe asn ala trp phe pro glu ala lys gln ala leu ala asp ile ala
Glu phe ala thr ser leu asp leu asp.
Additionally in accordance with a preferred embodiment of the present invention, the esterase protein is derived from the Acinetobacter strain A. calcoaceticus BD4.
Further in accordance with a preferred embodiment of the present invention, the esterase protein is derived from the Acinetobacter strain A. calcoaceticus BD13.
There is also provided in the present invention a method of forming and stabilizing an oil-in water emulsion, comprising:
a) adding esterase protein of 32.5 KD, found in association with the emulsan protein in the bacteria Acinetobacter, isolated from cell extracts from at least one strain of Acinetobacter, or recombinant preparations of said esterase protein isolated from esterase-producing vectors expressed in suitable hosts, or peptide fragments of said esterase protein produced in any of a variety of means, including proteolysis or genetic cloning;
b) adding water-soluble polysaccharide polymer of any source, including bacterial, plant and synthetic sources; or biopolymers such as polyanionic heteropolysaccharides.
Additionally in accordance with a preferred embodiment of the present invention, the method further comprises the step of adding a stabilizer or emulsifier.
In accordance with a preferred embodiment of the present invention, the method further comprises the step of adding emulsan or apoemulsan.
Moreover in accordance with a preferred embodiment of the present invention, the method further comprises the step of adding low molecular weight surfactants.